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Aspirin is chemically acetylsalicylic acid. It is hence called as salicylate drug and is very commonly used as an analgesic, antipyretic and anti-inflammatory. On the other hand aspirin shows an antiplatelet action by the inhibition of thromboxane production. Thromboxane usually patches the damaged walls of blood vessels by binding the platelets together. So by inhibition of thromboxane production, aspirin prevents the platelet patches from becoming larger and thus preventing these patches from blocking blood flow. Aspirin is used as a long-term drug at reduced doses for preventing strokes, cardiac attacks and clotting in persons with higher risk of blood clot formation (Lewis et al., 1983). According to a study, aspirin at low dose reduces the risk of heart attack after the previous one. Aspirin suppresses prostaglandins & thromboxanes by inactivating the enzyme cyclooxygenase irreversibly (Julian et al., 1996)
In this practical, pharmacokinetics of aspirin was studied by using HPLC, platelet aggregometry and calorimetry techniques.
(i) The aim of the practical was to study the pharmacokinetics of aspirin taken orally along with and without sodium bicarbonate.
(ii) To study the relationship between urinary pH and salicylic acid excretion.
(iii) To observe the effect of aspirin on platelet aggregation.
Materials and Methods:
Subjects were divided into 2 groups:
(i) those who ingested 600 mg aspirin
(ii) those who ingested 600 mg aspirin along with 10 g sodium bicarbonate
10-20ml of whole blood sample was taken from the subjects at 0 h (prior to taking aspirin), 1 h, 2 h, 4 h, 6 h, 8 h and 24 h after ingesting aspirin. At each point of time plasma was separated and platelet aggregation was studied. Later, by HPLC, ASA and salicylic acid levels in the plasma were quantitatively analysed.
Urine sample of each subject was collected at same time intervals as blood samples. The volume and PH of each urine sample were measured and noted down. Salicylate concentration in urine was determined by colorimetric method.
Platelet Aggregation Studies:
Platelet aggregation studies were carried out only at 0, 2 and 4 hours. Platelet aggregometry was carried out by the demonstrator. PPP was retained for HPLC analysis.
HPLC Determination of ASA and SA in Plasma:
Preparation of Standard Curves for ASA and SA:
ASA stock solution (0.2mg/ml)
SA stock solution (0.5mg/ml)
Phenacetin solution (0.5mg/ml)
Standard curves were prepared in duplicate as following:
Volume of water (µl)
Volume of ASA (µl)
Volume of SA
The values in brackets are final concentrations (mg/ml).
Extraction for Standard Curve and Samples:
Samples were vortex mixed. 500µl of each plasma sample was pipette into a test tube. Then for standards and samples 50µl of 0.5mg/ml phenacetin solution and 60µl of 1.0M HCL was added. Vortex mixed and 5 ml of diethyl ether was added and capped. This solution is mixed for 15 minutes
on a rotary mixer and centrifuged for 5 minutes at 3000rpm. The upper ether layer was transferred into a fresh test tube using Pasteur pipette and was evaporated. HPLC was carried out by the demonstrator.
Determination of Salicylate Levels in Urine:
Seven tubes were labelled as 0, 0.01, 0.02, 0.05, 0.1, 0.25, 0.5mg/ml. These are sodium salicylate standard solutions.
Urine samples were vortex mixed and 1ml of urine or standard sodium salicylate solution was taken into test tubes. To these tubes 5ml ferric nitrate solution was added and capped. Mixed using vortex mixer and absorbance was read at 525nm.
Platelet Aggregation (5µM ADP)
% Platelet Aggregation
Platelet Aggregation (1.5 Mm Arachidonic acid)
% Platelet Aggregation
HPLC Results of plasma samples:
Standard curve used: y =14.86x -0.007 (see graph 1)
Y= area ratio SA/PH (area of salicylic acid peak divided by area of phenacetin)
X= Salicylate concentration (mg/ml)
From equation of standard curve, salicylate concentration for each samples at different times were found out
SA/PH area ratio
Salicylate Concentration (mg/ml)
8.34 x 10-3
2.83 x 10-3 *(was flawed)
7.87 x 10-3
3.63 x 10-3
Graph 1: Standard curve of HPLC
Graph 2: Salicylate vs time standard curve
Graph 3: Area under curve (AUC)
Peak Plasma Concentration, Cmax= 0.009 mg/ml
AUC= A1 + A2 + A3 +A4 + A5
= 0.04 + 0.086+0.0168+0.0114 + ( Clast / Kel)
Kel = 0.15 per hour (From slope of Plasma conc v/s time plot on semi log graph)
Clast = last plasma concentration = 3.63 x 10 -3 mg/ml
AUC= 0.1772 mg/ml hours
Half life of SA from plasma = 4.62 hours (0.693 /kel)
Ka= 0.0014 per hour (from extrapolation of plasma concentration v/s time curve)
Standard curve for Sodium Salicylate:
y= 2.08x + 0.01
y= absorbance measured at 525nm
x= salicylate concentration
Salicylate (SA) concentration was found for each sample at different times, then converted it to amount of salicylate by multiplying the SA concentration by volume measured at that time
Then, Excretion rate = Amount of salicylate / Time period
abs. (at 525 nm)
Salicylate conc. (mg/ml)
Excretion rate (mg/h)
Cumulative Amount of Drug Excreted (mg)
Renal Clearance = Amount excreted in urine/ AUC
= 265.52 mg / 0.1172 mg/ml hours
= 1498 ml/hour
= 25.0 ml/min
Kel, obtained from slope excretion rate v/s time plot of urine= 0.343
Half life of SA from urine = 2.01 hours
Graph 4: Cumulative amount of drug excreted versus time was plotted
In the stomach very less amount of aspirin is ionized since it is weakly acidic and the environment in the stomach is also acidic. Due to this reason absorption of acetylsalicylic acid is delayed at high doses and takes up to eight to twenty four hours after ingestion. As aspirin moves into intestine it gets absorbed rapidly because of the increased pH in the intestine. This absorption is facilitated to further extend by the increased surface area of the intestine. In regard to the absorption of aspirin, it is absorbed very slowly when overdosed and the levels of aspirin in the plasma rises even till 24 hours from the time of administration (Levy and Tsuchiya, 1972). This is what we were trying to do study in this practical by giving sodium bicarbonate to increase the absorption of aspirin, to some extent in the stomach and to a larger extent in the intestine. The underlying mechanism of administering aspirin along with sodium bicarbonate is that, sodium bicarbonate creates an increased pH environment for aspirin to get absorbed at a faster rate and thus affecting platelet aggregation in turn. Almost fifty to eighty percent of salicylic acid is protein bound present in blood whereas the remaining is present in an active and in a state of ionization. The extent of protein binding depends on the concentration. As the binding sites get more and more saturated the amount of loose salicylic acid increases and in turn the toxic effects of the drug increases. The Vd of salicylic acid is 0.1-0.2 per kilogram. In high acid conditions the tissue entering capacity of salicylic acids increases and hence the Vd increases. Almost eighty percent of the drug metabolizes in liver. When aspirin is given in large doses, kinetics of the drug shift from 1st order to 0th order and metabolic pathways reaches a saturation point and hence urinary excretion is of increasing importance (Levy and Tsuchiya, 1972).
Graph 5: Graph showing percentage of platelet inhibition in all the four volunteers.
Volunteers I and IV ingested aspirin along with sodium bicarbonate and whereas volunteers II and III ingested only aspirin and it could be inferred from the results that sodium bicarbonate showed obvious effect on the percentage of platelet inhibition. Volunteers I and IV who were administered with aspirin along with sodium bicarbonate showed 100% and 73% inhibition respectively and at t=4 hours it is around 20% for volunteer I and was flawed for volunteer IV. This indicates that sodium bicarbonate decreased the effect of aspirin at t=4 hours whereas in both the volunteers at t=2 hours, there was no significant effect on percentage platelet inhibition because of aspirin being at utmost effective level. In case of volunteers II and III aspirin showed a significant effect on the percentage of platelet inhibition. For volunteer III at t=2 hours platelet inhibition percentage was around 60% and at t=4 hours it was around 85% indicating that there was high absorption of drug and increase in platelet aggregation must be due to increase in plasma concentration from time t=2 to t=4. But in case of volunteer II the values were flawed. This might be due to some sort of contamination during the procedure or might be human error in collecting the blood samples.
PRP was used in performing the platelet aggregation technique. To this PRP, ADP/Arachidonic acids were added at required concentrations which act as agonists. When the agonist is added, platelets get aggregated. The starting response of aggregation process is because of the addition of ADP or it may be because of the internal ADP being released which is present inside the platelets. Usually the acetylsalicylate blocks aggregation when the agonist doses are less but can't at higher doses. The aggregometry results shows that at t=2 hours percentage of platelet aggregation inhibiton was lower compared to that of at t=4 hours. This might be because of the absorption of aspirin into the body and thus causing inactivation of COX enzymes. This was made clearer with the result that adding of arachidonic acid, substrate for COX enzyme did not show aggregation due to blockage of the pathway.
Even though it is well established that aspirin inactivates COX-1 enzyme irreversibly through acetylation, anti-inflammatory effect through prostaglandin blockage and anti-thrombotic effect through Thromboxane A2 blockage, there are many other factors which affect the aggregation. Platelet inhibition usually reaches to highest peak with two hours of administration of aspirin. With the more and more impairment of platelet aggregation, there will be an increased number of platelets rushing from bone in order to maintain haemostatic environment (Schafer, 1999). ADP is comparatively a weak agonist and it has two receptors for it on platelet surface. These receptors are P2Y1 and P2Y12 and when these get activated they mobilize calcium and thus cause platelet aggregation (Michelson, 2007).